Abstract

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type‐specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation‐inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine–glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.

Introduction

Squamous cell carcinomas (SCCs) are lethal malignancies arising from the stratified epithelia of skin, esophagus, cervix, and the head and neck. Genomic analysis of SCCs identified genomic amplification of TP63 in up to 30% of tumors, with overexpression of its mRNA in the majority of SCCs [1], [2], [3], [4].

TP63, a p53 homolog, is a master transcriptional regulator of epithelial development and maintenance [5], [6], [7], [8], [9], [10], [11], [12]. Deletion of TP63 during embryogenesis resulted in the absence of skin and limbs [5], [6]. TP63 contains two different promoters to drive two distinct isoform classes either with or without N‐terminal transactivation domain, TAp63 and ΔNp63, respectively [9], [10], [11]. In addition, both TAp63 and ΔNp63 have three variants with different C‐termini (α, β, and γ) generated by alternative splicing [9], [10], [11]. ΔNp63α is the prominent isoform in adult epidermis [7], [13], [14], where it contributes to self‐renewal ability or stemness [[5], [6], [7], [8],[15], [16], [17]]. Of note, pharmacological inhibition of ROCK, a differentiation‐inducible serine/threonine kinase [18], [19], resulted in induction of continuous proliferation ability with elevated ΔNp63α expression in keratinocytes [20].

In the context of cancer, the major isoform of p63 detected in SCC is also ΔNp63α [21], [22], [23], [24], [25]. ΔNp63α functions as a transcriptional regulator of different gene subsets [13], [26], [27]. Forced expression of ΔNp63α was sufficient to drive proliferation and tumorigenesis [24], while inducible genetic deletion of TP63 blocked chemical‐induced SCC formation [23], underscoring ΔNp63α as an oncogenic transcription factor in SCCs. Although the role of ΔNp63α has been established, the process controlling ΔNp63α nuclear transport remains poorly understood.

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm [28], [29], [30], [31], [32], [33]. NPCs consist of multiple copies of roughly 30 different proteins known as nucleoporins (NUPs). About one‐third of NUPs, including NUP62, contain unstructured phenylalanine–glycine (FG) repeats, which form a soft and flexible cobweb composed of ~200 intrinsically disordered polypeptide chains, mediating selective nucleocytoplasmic transport [34], [35]. Recently, new evidence suggests that NPC composition varies among different cell types and tissues, and this heterogeneity is required for distinct biological processes such as muscle development, neurogenesis, and embryonic stem cell pluripotency [36], [37], [38], [39], [40], [41]. Importantly, single NUP mutation resulted in developmental defects and diseases [42], highlighting the significant role of NUPs in maintaining tissue homeostasis. However, whether and how NUPs are involved in the biology of stratified squamous epithelia remains largely unknown.

Here, we demonstrate that the expression of NUP62, one of FG‐NUPs, is abundant in stratified epithelia. Aberrant overexpression of NUP62 is evident in human SCCs. NUP62 expression is required to maintain undifferentiated status of SCCs through regulating ΔNp63α nuclear transport. Mechanistically, pro‐differentiation ROCK kinase negatively regulates this ΔNp63α nuclear transport by limiting the interaction between ΔNp63α and NUP62 through phosphorylation of the FG regions of NUP62.

Results

Identification of NUP62 as a novel differentiation‐preventing factor in SCC

To explore the expression profile of NUPs in healthy human tissues, we re‐analyzed RNA‐seq data from The Human Protein Atlas (see Materials and Methods). The transcript levels of six NUPs (NUP107, NUP85, NUP62, NUP54, NUP188, and TPR) were higher in representative stratified epithelia, skin, and esophagus relative to other NUPs (Fig 1A). Among them, NUP62 was the only gene displaying increased expression during induction of the epidermal lineage (Fig 1B). To examine expression profile of NUP62 in skin tissue, we performed immunofluorescent confocal microscopic analysis using healthy human skin sample (Fig 1C). We found that NUP62 was expressed in the undifferentiated layer, indicating NUP62 is expressed in the progenitor population.

Western blot analysis of each NUP in SCC cells at 72 h after siRNA‐mediated knockdown.

Plot showing the correlation between average effect of NUP depletion on SAS cell growth and differential gene expression (normal tissue versus HNSCC from TCGA). NUP62 was highlighted in orange. TP63 was marked in purple as positive control for oncogene. Data show mean from two independent experiments (n = 2).

Summary of correlation coefficient between each NUPs mRNA and epidermal differentiation genes (SPRR1B and IVL) in head and neck SCC from TCGA.

qRT–PCR analysis of genes associated with epidermal differentiation after individual depletion of each NUPs. Transcripts of each gene with scrambled siRNAs is considered 1.0. Data show mean ± SD (n = 3; NUP62i) or mean (n = 2; others). P values are based on one sample t‐test with *indicating P < 0.05.

In order to determine the expression profile of NUPs in SCC, we compared the expression of above six NUPs mRNAs in normal versus tumor samples using data from both the RNA‐sequencing from The Cancer Genome Atlas (TCGA) and the cDNA microarray data from Gene Expression Omnibus (GEO; series GSE7410). We found that NUP62 was the top gene with consistent overexpression in primary SCC samples from both head and neck (Fig 1D) and cervix (Fig 1E).

To dissect the role of the above six NUPs in SCC growth, we individually silenced expression of each NUP (Figs 1F and EV1A), and identified that NUP62 is required for the proliferation of SCC cells (Figs 1G and EV1B). Next, we investigated whether other highly expressed NUPs in SCCs (NUP62, NUP107, NUP85, and NUP54) regulate epidermal differentiation. We queried TCGA datasets to determine co‐expression profiles among these NUPs and epidermal differentiation genes such as IVL and small proline‐rich protein 1B (SPRR1B). NUP62 was the only NUP showing an inverse correlation with both IVL and SPRR1B (Pearson Score < −0.3; Fig 1H). Moreover, NUP62 depletion activated these epidermal differentiation genes, a result not observed in any other NUPs (Fig 1I). Taken together, these data strongly suggest that NUP62 has cell type‐specific function to prevent epidermal differentiation induction in SCCs.

A. Levels of transcripts in cells depleted of the indicated NUPs were measured by qRT–PCR. Transcript levels of each NUPs with scrambled siRNAs are considered 100%. Data show mean from two independent experiments (n = 2).

B. Relative viable number of UMSCC1 and SAS SCC cells depleted of each NUP after siRNA treatment compared to transfection with scrambled siRNA (Control). Value of cells with scrambled siRNAs is considered 100%. Data show mean from two independent experiments (n = 2).

NUP62 is required for the proliferation and prevent of differentiation of SCC cells

To study the biological role of NUP62 in SCC, we silenced NUP62 in a number of cell lines from cervical SCC (CSCC) and head and neck SCC (HNSCC) by siRNA‐mediated knockdown (Fig 2A). Notably, silencing of NUP62 strongly inhibited the proliferation of various types of SCC cells (Fig 2B). Several cell lines, including ME‐180, UMSCC1, and Ca9‐22, barely grew in the absence of NUP62. Similarly, shRNA‐mediated NUP62 depletion resulted in significant reduction in short‐term cell proliferation and foci formation of SCC cells (Fig EV1C–E).

As lack of differentiation is a hallmark of SCC, we measured levels of molecular markers associated with squamous cell differentiation such as SPRR1B, IVL, filaggrin (FLG), and loricrin (LOR). Silencing NUP62 enhanced the expression of these genes (Fig 2C). Interrogation of TCGA RNA‐seq datasets showed that expression levels of these differentiation related genes are inversely related to levels of NUP62 (Fig 2D). Taken together, these data strongly suggest that NUP62 is a functional requisite for SCCs.

Transcriptome analysis of NUP62‐regulated genes and processes

To delineate the SCC‐associated functions of NUP62, cDNA microarray was performed in UMSCC1 cells comparing the silencing of the protein versus control cells. Loss of NUP62 altered the expression of 2,019 genes compared to control (twofold change, Fig 3A). Activated genes after NUP62 silence were enriched in Gene Ontology terms related to phosphorylation, regulation of localization, Rho guanine nucleotide exchange factors (GEF), negative regulation of cell proliferation, epithelial cell differentiation, and regulation of transport (Fig 3B). Interestingly, gene set enrichment analysis (GSEA) identified a strong correlation between down‐regulated genes in NUP62‐silenced cells and those in p63‐depleted SCCs (GSE88833; Fig 3C). As NUP62 depletion did not affect the level of ΔNp63α transcript per se (Fig 3D and E). We next explored alternative mechanisms underlying this molecular association.

qRT–PCR analysis of p63 and NUP62 mRNA in various SCC cell lines depleted of NUP62. Expression levels of cells treated with scrambled siRNAs is considered 100%. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05 and **P < 0.01.

Western blot analysis of ΔNp63α in SCC cells after siRNA‐mediated NUP62 depletion.

NUP62 regulates ∆Np63α nuclear transport in SCCs

To examine whether NUP62 affects p63 localization, we quantified the amount of nuclear and cytoplasmic ∆Np63α using confocal microscopy. Attenuated nuclear ΔNp63α level was detected in NUP62‐depleted SCC cells (Fig 4A and B). Concordantly, nuclear‐cytoplasmic fractionation demonstrated that ΔNp63α abundance in the nuclear fraction was less in the NUP62 silenced SCC cells compared to the control cells (Fig 4C).

Transcript amounts of TAp63 targets were measured by qRT–PCR. Transcripts of each gene with scrambled siRNAs are considered 1.0. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test. n.s. indicates not significant.

qRT–PCR analysis of IVL and SPRR1B mRNA in SCC cells treated with Y27632. Expression levels of cells with DMSO is considered 100%. Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05 and **P < 0.01.

B, C SCC cell lines under Y27632 treatment (10 μM) were subjected to MTT assay (B) and foci formation assay (C). Number of colonies of control cells is considered 100%. Data show mean ± SD from three independents (n = 3). P values are based on two‐tailed t‐test (B) or one sample t‐test (C) with *indicating P < 0.05.

D. Proliferation of SCC cells expressing Δ3‐ROCK1 and the effect of Y27632 (10 μM) were analyzed by MTT assay. Data show mean ± SD from three independents (n = 3). P values are based on unpaired two‐tailed t‐test with *indicating P < 0.05.

Phosphorylation of FG domain of NUP62 mediates the interaction between ΔNp63α and NUP62

In order to elucidate ΔNp63α nuclear transport system, we first addressed whether it involves nuclear transport receptors (NTRs). Since nuclear localization signal (NLS), an amino acid sequence consisting of positively charged lysines (K) or arginines (R), tags proteins for NTR‐mediated traffic [57], we searched for nuclear localization signal (NLS) on ΔNp63α. Based on NLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi), two potential NLSs was found in ΔNp63α (Fig 6A). To explore the biological functions of those candidate NLSs, we generated full‐length ΔNp63α (ΔNp63αWT), NLS1‐deleted ΔNp63α (ΔNp63α∆NLS1), and NLS2‐deleted ΔNp63α (ΔNp63α∆NLS2), all of which were fused with GFP at the C‐terminus (Fig 6A). Quantification by fluorescent confocal microscopy showed that the vast majority of ΔNp63αWT and ΔNp63α∆NLS2 localized in the nucleus (Fig 6B). In contrast, ~30% cells expressing ΔNp63α∆NLS1 exhibited signals in both the cytosol and nucleus (Fig 6C). Importantly, NLS1 sequence on ΔNp63 showed high similarity with validated NLS of both p53 and p73 (Fig 6D) [58], [59], [60], suggesting that ΔNp63 nuclear import is mediated through NLS1.

Figure EV4.Expression profile of ΔNp63 isoforms and KPNB1 in SCC cells, and potential ROCK targeting region on FG of NUP62

Western blot analysis of ΔNp63α and KPNB1.

Evolutionarily conserved phosphorylation sites in FG domain of NUP62.

Quantification of Western blots results showing ROCK1 activation resulted in elevated phosphorylation of FG of NUP62, while Y27632 reduced this phosphorylation. The relative amount of phosphorylation was quantified by ImageJ using pull‐downed FG amounts as an internal control and normalized further to this level in control (CTL). Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05. n.s. indicates not significant.

Diagram of mutations made in FG domain.

Source data are available online for this figure.

NUP62 harbors FG repeated region in N‐terminus required for nucleocytoplasmic regulating factors, and coiled‐coil (CC) domain in C‐terminus to bind with NUP54 [61] (Fig 6G). To explore the basis for the functional relationship between ROCK1 pathway and NUP62‐dependent ΔNp63α nuclear transport, we constructed NUP62 fragments and performed immunoprecipitation (IP) analysis to determine p63‐interacting regions on NUP62 (Fig 6G). Of note, ΔNp63α preferably bound to the FG region (Fig 6H). Analysis of PhosphoSitePlus (see Materials and Methods) revealed that the FG domain of NUP62 includes potential phosphorylation sites, S2 and T20 (Fig 6H), which are highly conserved (Fig EV4B). Next, in order to examine whether the ROCK1 pathway phosphorylates FG domain, Δ3‐ROCK1 was co‐expressed with the FG fragment, and cell lysates were immuno‐precipitated with anti‐GFP antibodies and then immunoblotted with phosphor‐serine/threonine antibody (Fig 6I). Co‐expression of the FG domain fragment and Δ3‐ROCK resulted in a prominent enrichment of FG domain fragments having elevated phosphorylation (Figs 6I and EV4C). Importantly, this phosphorylated FG domain fragment had decreased interaction with ΔNp63α (Fig 6J). To explore further the biological relevance of the phosphorylation on FG domain of NUP62, we generated an alanine replaced mutant (Amt) NUP62 (Fig EV4D). Importantly, point mutations of the phosphorylation sites (S2A and T20A) enhanced the interaction between NUP62 and ΔNp63α (Fig 6K), suggesting the phosphorylation of FG domain of NUP62 decreased the interaction between NUP62 and ΔNp63α.

ΔNp63α nuclear transport is mediated by the phosphorylation of FG domain of NUP62

C, D Various SCC cell lines expressing either WT or Amt NUP62 were subjected to foci formation assay (C). Number of colonies from cells expressing WT NUP62 is considered 100% (D). Data show mean ± SD from three independent experiments (n = 3). P values are based on one sample t‐test with *indicating P < 0.05.

Discussion

In this study, we showed novel trafficking regulation by NUP62 in human SCCs. Importantly, NUP62 regulated ΔNp63α nuclear transport to prevent epidermal differentiation in SCCs. ROCK pathway inhibited the ΔNp63α nuclear transport by reducing interaction between ΔNp63α and NUP62 through the phosphorylation of the FG domain of NUP62.

Heterogeneity of NPC and the cell type‐specific properties of some pore components have been reviewed [42]. Based on in silico analysis of NUPs transcripts across normal tissues, NUP62 expression levels were prominent in stratified epithelia tissues. We further determined that basal progenitor cells tend to express NUP62 in human skin tissue. In addition, SCC showed excessive levels of NUP62 compared to healthy tissues. We validated the function of NUP62 to maintain proliferative activity and suppress differentiation of SCCs, highlighting the cell type‐specific role of NUP62 to regulate the cellular state of epidermal progenitor cells. Interestingly, Toda et al [63] recently uncovered the cell type‐specific role of NUP153 in the control and maintenance of neural progenitor cells through bimodal transcriptional regulation. Present study demonstrated the role of NUP62 in SCC, however, how those NUPs cooperate in the regulation and maintenance of cell identity in different tissues and other types of cancer cells will be of interest.

We found that NUP62 regulated ΔNp63α nuclear transport to maintain cellular undifferentiated status of SCCs. Importantly, p63 can be overexpressed including by genomic amplification, and the expression level of ΔNp63α is a crucial clinical–pathological parameter affecting malignant potential of primary SCCs [1], [2], [3], [4]. Furthermore, previous studies demonstrated the mechanistic action of ΔNp63α in SCC [10], [11]. For example, ΔNp63α inhibits the activity of TAp73 [64] via transcriptional inhibition and, to a lesser extent, direct interaction between both proteins [65]. Therefore, attenuation of ΔNp63α nuclear transport can serve as a new strategy to compromise its functions.

Nuclear transport receptors including KPNB1 are suggested to aid the shuttle of macromolecules larger than ~40 kDa. These NTRs bind to FG‐NUPs, allowing the transport factors to cross NPC [66]. Here, we identified a candidate functional NLS (containing KKRR) on ΔNp63α, which shows similarity with validated NLS on both p53 and p73 [58], [59], [60]. Previous studies demonstrated that destruction of KKRR on p53 and p73 almost completely blocked their nuclear accumulation [58], [59], [60]. In contrast, the effect of mutation of KKRR of ΔNp63α was moderate in nuclear blockage. ΔNp63α forms hetero‐tetramers among p53 family members [10], [11] and makes subcomplexes with cofactors such as p300 [67], which determine specificity and selectivity of downstream targets of ΔNp63. Therefore, NLS on interacting proteins may represent ΔNp63α nuclear traffic to some extent. Identification of the p53 family protein complexes will enhance our understanding of the ΔNp63α nuclear traffic system as well as selectivity of downstream targets of ΔNp63α.

In addition, this study showed that the interaction between ΔNp63α and FG domain of NUP62 is required for ΔNp63α to enter the nuclear space in SCCs. Importantly, ROCK1‐dependent phosphorylation on FG domain resulted in attenuated interaction with ΔNp63α as well as the nuclear transport ability. The ROCK1‐specific inhibitor Y27632 was demonstrated to transform epithelial cells into continuously proliferating cells by increasing progenitor cell factors such as ΔNp63α [20], suggesting that the ROCK pathway inhibits the self‐renewal ability by suppressing ΔNp63α expression. Previously, Kosako et al reported that ERK phosphorylation of the repeat region of FG of Nup50 reduced its affinity for particular NTRs, resulting in impaired nuclear migration of the NTRs [47]. Taken together, these findings suggest that transport ability and selectivity of NPC are controlled by kinase‐dependent phosphorylation on the FG domain of FG‐NUPs.

In summary, we show that high expression of NUP62 contributes to preventing epidermal differentiation of SCCs originating from stratified epithelia. Mechanistic studies reveal that NUP62 maintains ΔNp63α entering NPC, whereas ROCK negatively regulates this process. Our results highlight cell type‐specific role of NUP62 controlling systematically ΔNp63α nuclear transport in SCCs. Our finding of convertible trafficking activity of NUP62 highlights the potential for therapeutic targeting of nuclear transport of this oncogene.

Cell proliferation assay

Cells were seeded into a 96‐well plate at 3,000 cells/well and cultured for the indicated time courses. Cell viability was assessed using the MTT (3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide) method. In brief, 10 μl of 12 mM MTT solution was added into each well followed by 3‐h incubation, which was stopped by adding 100 μl of STOP solution (2% acetic acid, 16% SDS, 42% DMF). Samples were mixed thoroughly and measured at 570 nm for absorbance.

Foci formation assay

Squamous cell carcinoma cells were seeded into 6‐well plates at 1,000 cells/well and cultured for 10 days, followed by fixation, staining with crystal violet and photographing the cells using LAS4000 (Fujifilm, Aichi, Japan). For quantification, the number of colonies (> 0.1 mm2) was determined using Multi Gauge Ver3.0 (Fujifilm).

Microscopic analysis using SCC cells

Cells on coverslips were incubated under indicated conditions. Cells were fixed for 10 min in 4% paraformaldehyde in PBS, then permeabilized with 0.3% Triton X‐100 in PBS for 3 min at room temperature. Coverslips were incubated with indicated primary antibody for 2 h. Coverslips were washed three times and incubated with Alexa Fluor‐conjugated secondary antibody (Life Technologies) for 1 h. After three washes, samples were mounted onto coverslips using Pro‐Long Gold Antifade reagent (Life Technologies) and were examined by confocal microscopy (FluoView® FV10i, Olympus, objective ×60/1.2). Nuclear ΔNp63α signals were quantified with FV10i (Olympus).

cDNA vectors and siRNAs

The NUP62 fragment vectors have been described previously [68]. Constructs for overexpressing NUP62 were made by cloning cDNA derived from HaCaT into pLEX (Open Biosystems) using NotI/AgeI with C‐terminal FLAG tag with the following primers: NUP62‐F:5′‐ACAGTGGCGGCCGCGCCACCATGAGCGGGTTTAAT‐3′; NUP62‐R: 5′‐GGCGACCGGTTTACTTGTCGTCATCGTCTTTGTAGTCGTCGTCAAAGGTGATCCG‐3′. NUP62 CDS harboring mutations (S2A, T20A) was generated with the following primers: S2A‐F1: 5′‐ACAGTGGCGGCCGCGCCACCATGGCCGGGTTTAAT‐3′; T20A‐R: 5′‐CTTTGCAGCGCCAAAC‐3′; T20A‐F: 5′‐GTTTGGCGCTGCAAAG‐3′; Mt3end‐R: 5′‐GGCGACCGGTTTACT. Two fragments were generated by PCR with S2A‐F/T20A‐R and T20A‐F/Mt3end‐R. After purification, these fragments were mixed and subjected to PCR using S2A‐F/Mt3end‐R. Constructs for overexpressing MYC‐tag‐fused Δ3‐ROCK1 were made by cloning cDNA derived from HaCaT into pLEX (Open Biosystems) using XhoI/AgeI sites. CDS‐Seq was reported previously (National Center for Biotechnology Information (NCBI), see URL). The siRNAs targeting human nucleoporins are listed in Table 2, and scramble siRNA (D‐001210‐01) was purchased from Thermo Scientific.

Transfections, viral particle production, and infection

DNA and siRNA transfections were performed using Lipofectamine 2000 and Lipofectamine RNAiMAX (Life Technologies), respectively. Lentiviral particles were produced with the MISSION Lentiviral Packaging System (Sigma‐Aldrich). SCCs cells were transduced with the lentiviral particles in the presence of 8 μg/ml polybrene (Sigma‐Aldrich) for 48 h.

cDNA preparation and quantitative Real‐time RT–PCR assay

We used 500 ng RNA for cDNA preparation using ReverTra Ace® qPCR RT Master Mix (TOYOBO). Quantitative real‐time RT–PCR was performed by SYBR® Premix Ex Taq™ II (Takara) in a Thermal Cycler Dice® Real Time System (Takara) according to the manufacturer's instructions. The relative mRNA expression level of target genes was calculated using GAPDH as a loading control. The primers are listed in Table 3.

Microscopic analysis

Cells on coverslips were overexpressed Δ3‐ROCK1 and then further incubated with either Y27632 or the CRM1 inhibitor for 24 h. For ΔNp63 staining, cells were fixed for 10 min in 4% paraformaldehyde in PBS, then permeabilized with 0.2% Triton X‐100 in PBS for 20 min at room temperature. Permeabilization was proceeded before fixation in RanBP2/Flag dual staining [68]. Coverslips were incubated with indicated primary antibody for 2 h. Coverslips were washed three times and incubated with Alexa Fluor‐conjugated secondary antibody (Life Technologies) for 1 h. After three washes, samples were mounted onto coverslips using Pro‐Long Gold Antifade reagent (Life Technologies) and examined using confocal microscope (FluoView® FV10i, Olympus, objective ×60/1.2). Nuclear p63 signals were quantified with FV10i (Olympus).

Statistical analyses

Following assays: cell proliferation assay, anchorage‐dependent colony formation assay, and real‐time RT–PCR analysis were performed in triplicates (n = 3) and independently replicated 2–4 times. Data are the average of independent experiments (n = 2 or 3). Unpaired two‐tailed t‐test was performed by GraphPad Prism in the statistical analysis of the following assays: cell proliferation assay, anchorage‐dependent colony formation assay, and real‐time RT–PCR analysis. P < 0.05 was considered statistically significant. When control group is considered as 100%, one sample t‐test was performed using GraphPad QuickCalcs (see URLs).

Conflict of interest

Expanded View

Acknowledgements

We thank Takayuki Dowaki, Kie Sakai, and Ryota Tsukahara for technical support. This work was funded by an Infiniti Grant and in part by Grants‐in‐Aid for scientific research (JSPS KAKENHI Grant Number 17K16332) and (JSPS KAKENHI Grant Number 80292423) from the Japan Society for Promotion of Science. This research was supported by the grant provided by The Ichiro Kanehara Foundation. This work was also supported by World Premier International Research Center Initiative (WPI), MEXT, Japan.

Funding

The Ichiro Kanehara Foundationhttp://dx.doi.org/10.13039/501100003837

Japan Society for the Promotion of Science London (JSPS)http://dx.doi.org/10.13039/50110000064617K16332

Japan Society for the Promotion of Science (JSPS)http://dx.doi.org/10.13039/50110000169180292423